The teleost acute-phase inflammatory response and caspase activation by a novel alarmin-like ligand.

This study tested the hypothesis that NCAMP-1 has alarmin-like properties and activates the caspase-1-binding site in cells of the teleost bone marrow (equivalent). In mammals, alarmins have been studied extensively; however, in teleosts, little is known about their identity and functions. Similar to alarmins, NCAMP-1 has a broad spectrum of bacteriolytic activity. NCAMP-1 is constitutively present in CF serum, and levels were increased following infection with Edwardsiella ictaluri Binding to AK cells was determined with rNCAMP-1 and an anti-His-tag antibody. In vitro treatment of AK (bone marrow equivalent) or spleen cells with rNCAMP-1 increased the IL-1ß message three- to fivefold at 3 h, 6 h, and 9 h post-treatment. The association of NCAMP-1 with the activities of alarmin ATP and the acute inflammatory response was demonstrated by NCAMP-1-induced P2X7R pore opening and YO-PRO-1 cellular influx. The association of NCAMP-1 binding with inflammasome activation was demonstrated by NCAMP-1 activation of the caspase-1-binding site for tetrapeptide Z-YVAD-FMK. In competition assays, this tetrapeptide competitively inhibited subsequent binding by the pan-caspase substrate tripeptide FAM-VAD-FMK. Lymphocyte-like cells from the spleen were 16%+, and epithelial cells were also positive for NCAMP-1. IHC staining and confocal microscopy confirmed the cytosolic existence of NCAMP-1 in lymphoreticular tissue and IL-1ß in AK cells. CF T cell lines G14D and 28S.3 expressed NCAMP-1 in the cytosol and in storage granules. These studies strongly suggested that NCAMP-1 is an alarmin-like ligand with similar but distinct activities to those of ATP and HMGB-1.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish.

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

Cellular expression and antimicrobial function of a phylogenetically conserved novel histone 1x-like protein on mouse cells: a potential new class of pattern recognition receptor.

A H1x-like protein (i.e., NCAMP-1) is expressed on the membrane and in GEs from fish NK-like cells. In the present study, we identify the imprinting control region mouse NCAMP-1 ortholog using NCAMP-1 polyclonal antibodies and mAb. Polychromatic flow cytometry revealed NCAMP-1 expression on PBLs (Gr-1(+) PMNs were 21.1% NCAMP-1(+); DX-5(+) NK cells were 12.2% NCAMP-1(+)), mesenteric LN cells (CD11c(+) DCs were 23.2% NCAMP-1(+); Gr-1(+) PMNs were 24.8% NCAMP-1(+); CD21(+) B cells were 17.8% NCAMP-1(+)), and splenocytes (CD11c(+) were 39.6% NCAMP-1(+); Gr-1(+) PMNs were 40.9% NCAMP-1(+); DX-5(+) NK cells were 24.3% NCAMP-1(+); CD21(+) B cells were 28.5% NCAMP-1(+)). Western blot analysis using pNCAMP-1 and GEs from RAW 264.7 cells produced a 32-kDa signal. GEs from RAW 264.7 cells produced a significant reduction in Escherichia coli CFU. This antimicrobial killing activity was inhibited by pretreatment of the extract with (polyclonal) anti-NCAMP-1. Treatment with preimmune serum did not reduce bacterial cell killing. Confocal microscopy using NCAMP-1 and LAMP-1 mAb demonstrated that NCAMP-1 was located on the membrane and in cytosolic vesicles of RAW 264.7 cells and did not appear to colocalize with LAMP-1. NCAMP-1 may participate as a bifunctional protein on cells. It is expressed on the membranes of phagocytic cells, NK cells, and APCs in mice as well as in the granules of macrophages. In phagocytic cells, NCAMP-1 may participate in a nonregulated exocytosis pathway of cellular secretion.

Role of nonspecific cytotoxic cells in bacterial resistance: expression of a novel pattern recognition receptor with antimicrobial activity.

Pattern recognition receptors (PRR) recognize invariant bacterial, viral, protozoan and certain synthetic ligands. PRR may be expressed as outer membrane (or endosomal) or cytosolic proteins and function to signal cell activation processes during inflammation responses. In the present study, a novel membrane receptor, NCC cationic antimicrobial protein-1 (NCAMP-1), is described that is expressed on nonspecific cytotoxic cell (NCC) membranes and is found in granule extracts from these cells. In recombinant form, full-length (amino acids 1-203) and truncated N (NT; amino acids 1-60) and C (CT; amino acids 116-203) terminal forms of NCAMP-1 had antibacterial activity against bovine, avian and lab strain Escherichia coli. Recombinant NCAMP-1-NT also killed the gram-negative fish pathogen Edwardsiella ictaluri. Maximal bacterial killing of a representative avian E. coli, APEC 3721, occurred at 60min post-treatment with 2microg/ml of rNCAMP-1-NT. Killing occurred by NCAMP-1-NT-induced alterations in the permeability of the bacterial cell wall. Polyclonal antibody anti-NCAMP-1 specifically neutralized the antimicrobial activity of recombinant NCAMP-1-NT against E. coli APEC 3751. Expression of NCAMP-1 as a NCC membrane protein was analyzed by flow cytometry using anti-NCAMP-1 monoclonal antibody 9C9. Merged images from immunofluorescence microscopy showed that NCAMP-1 and the NCC receptor protein (NCCRP-1) are co-expressed on NCC membranes. NCAMP-1 was identified in acetic acid granule extracts of NCC by Western blot analysis using polyclonal anti-NCAMP-1 and killing of E. coli by these extracts was specifically inhibited by this polyclonal. These data suggested that NCAMP-1 is a membrane protein and may participate in antibacterial innate immunity by granule exocytosis during inflammatory responses in teleosts.

Molecular characterization and expression of a granzyme of an ectothermic vertebrate with chymase-like activity expressed in the cytotoxic cells of Nile tilapia (Oreochromis niloticus).

We have identified the gene coding for a novel serine protease with close similarities to mammalian granzymes from nonspecific cytotoxic cells of a teleost fish Oreochromis niloticus. The genomic organization of tilapia granzyme-1 (TLGR-1) has the signature five-exon-four-intron structure shared by all granzymes and similar hematopoietic Ser proteases. Molecular modeling studies suggested a granzyme-like structure for this protein with four disulfide linkages and two additional Cys residues. The expression of this gene is found to be restricted to cytotoxic cell populations with a low level of constitutive expression when compared to similar granzymes in other teleost species. High levels of transcriptional activation of TLGR-1 with different stimuli suggested that this gene is highly induced during immune reactions. Triplet residues around the active site Ser of TLGR, which determines the primary substrate specificity of granzymes, differ significantly from that of other granzymes. Recombinant TLGR-1 was expressed in the mature and proenzyme forms using pPICZ-alpha vector in the Pichia pastoris expression system. Recombinant TLGR-1 was used to determine the primary substrate specificity of this protease using various synthetic thiobenzyl ester substrates. In vitro enzyme kinetics assays suggested a preference for residues with bulky side chains at the P1 site, indicating a chymase-like activity for this protease. These results indicate the presence of novel granzymes in cytotoxic cells from ectothermic vertebrates.

Nonspecific cytotoxic cells of teleosts are armed with multiple granzymes and other components of the granule exocytosis pathway.

Granzymes are members of the serine protease family and major components of cytotoxic granules of professional killer cells. Multiple granzymes have been identified from human and rodents with different substrate specificities. Although the significance of granzymes A and B in cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other granzymes may have either equal or greater importance in mediating cell death. Studies on the evolution of these closely related proteases were hindered by the lack of sequence and biochemical information of granzymes from "lower vertebrates." Here we report the generation of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester substrates verified its tryptase activity in full agreement with previous observations by sequence comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these cells. Evidence for additional granzymes with different substrate specificities in NCC was obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity assays. Searches of the catfish EST database further confirmed the presence of teleost granzymes with different substrate specificities. Granzyme activity measurements suggested a predominance of chymase and tryptase activities in NCC. Further proof that the granule exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts.

Constitutive expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in regulation of cell-mediated cytotoxicity.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main killer cell populations of the immune system. The mechanisms by which these cells recognize target cells vary considerably, while the effector molecules used to facilitate target cell death are highly conserved. The main pathways utilized by killer cells consist of granule exocytosis and those mediated by members of the TNF superfamily. Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in teleosts. We have previously demonstrated the expression of granzymes and Fas ligand in these cells. This is the first report of the expression of tumor necrosis factor-alpha in these killer cells. A cDNA coding for TNF was cloned and sequenced from NCC purified from Nile tilapia (Oreochromis niloticus). Factors regulating the transcriptional modulation of TNF in these cells were identified by RT-PCR analysis. The mature form of tilapia TNF was expressed as a recombinant protein and biological activities were analyzed. Using a cross-reacting anti-TNF polyclonal antibody, analysis of TNF expression suggested that tilapia NCC constitutively express the membrane-bound as well as secreted forms of TNF. Recombinant tilapia TNF effectively induced cytotoxicity in the mammalian cell line WEHI, although to a lesser extent compared to the murine TNF. Treatment with recombinant TNF protected NCC from activation-induced cell death. Recombinant tilapia TNF was also effective in upregulation of granzyme transcription in tilapia NCC. These data suggest that teleost TNF may play a role in diverse effector functions of cytotoxic cells from ectotherms, similar to the biological functions described for mammalian TNF.

Molecular cloning of cellular apoptosis susceptibility (CAS) gene in Oreochromis niloticus and its proposed role in regulation of non-specific cytotoxic cell (NCC) functions.

Cellular apoptosis susceptibility (CAS) gene is a homologue of the chromosome segregation gene (CSE) in yeast, involved in multiple cellular mechanisms associated with cell proliferation as well as cell death. CAS is highly expressed in proliferating cells but at a lower level in quiescent cells and tissues. Therefore it appears that CAS may play an important role in cancer development. We have previously identified CAS in tilapia non-specific cytotoxic cells (NCC) with a cross-reacting monoclonal antibody. Its expression was up-regulated in NCC in response to apoptosis regulatory factors. In the present report, the molecular cloning and expression of CAS in NCC is described, suggesting the importance of this protein in regulation of teleost immune functions. Furthermore, CAS expression is proposed as one of the mechanisms of regulation of activation induced programmed cell death (AIPCD) in these cytotoxic cells. As CAS expression is ubiquitous, we expect that these studies will help identify proliferating cells protected from apoptosis in additional tissues.

Molecular characterization of a novel pattern recognition protein from nonspecific cytotoxic cells: sequence analysis, phylogenetic comparisons and anti-microbial activity of a recombinant homologue.

Nonspecific cytotoxic cells (NCC) are the first identified and most extensively studied killer cell population in teleosts. NCC kill a wide variety of target cells including tumor cells, virally transformed cells and protozoan parasites. The present study identified a novel evolutionarily conserved oligodeoxynucleotide (ODN) binding membrane protein expressed by channel catfish (Ictalurus punctatus) NCC. Peptide fingerprinting analysis of the ODN binding protein (referred to as NCC cationic anti-microbial protein-1/ncamp-1) identified a peptide that was used to design degenerate primers. A catfish NCC cDNA library was used as template with these primers and the PCR-amplified product was sequenced. The translated sequence contained 203 amino acids (molecular mass of 22,064.63 Da) with characteristic lysine rich regions and a pI=pH 10.75. Sequence comparisons of this protein indicated similarity to zebrafish (51.2%) histone family member 1-X and (to a lesser extent) to trout H1. A search of EST databases confirmed that ncamp-1 is also expressed in various tissues of channel catfish as well as zebrafish. Inspection for signature repeats in ncamp-1 and comparisons with histone-like peptides from different species indicated the presence of multiple lysine based motifs composed of AKKA or PKK repeats. The novel protein was cloned, expressed in E. coli and the recombinant was used to generate rabbit anti-serum. The recombinant ncamp-1 bound GpC and CpG ODNs and was detected with homologous anti-ncamp-1 polyclonal antibodies. Western blots of NCC membranes using anti-ncamp-1 serum detected a 29 kDa protein. Binding competition experiments demonstrated that anti-ncamp-1 antibodies and GpC bound to the same protein on NCC. Two different truncated forms of ncamp-1 as well as the full-length recombinant protein exhibited anti-microbial activity. The present study demonstrated the expression by NCC of a new membrane protein that may participate in the recognition of bacterial DNA and as such participate in innate anti-microbial immune responses in teleosts.

Single base oligodeoxyguanosine upregulates Fas ligand release by nonspecific cytotoxic cells.

Nonspecific cytotoxic cells (NCC) are a type of teleost NK-like cell. In the present study a novel stimulus secretion model is described for catfish NCC utilizing single base oligodeoxyguanosine. Binding of guanosine 20-mers (dG20) to NCC up-regulated expression of cytosolic FasL detected by an anti-human FasL monoclonal antibody (mab). In vitro treatment of purified NCC with dG20 produced a 7-fold increase in expression of soluble Fas ligand (sFasL) after 3 h. Antibody binding to NCC was saturable and approximately 30-35% of total NCC were positive for sFasL expression. The teleost FasL equivalent produced programmed cell death of appropriate FasR positive targets. Supernatants from dG20 activated NCC produced hypoploidy and annexin-V binding by FasR bearing HL-60 cells. Treatment of activated supernatants with immobilized anti-FasL mab neutralized these activities. These studies demonstrated that an NK like cell (NCC) produces and secretes sFasL following binding by single base oligodeoxyguanosine.

Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells.

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with approximately 50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure-function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.

Identification of a scavenger receptor homologue on nonspecific cytotoxic cells and evidence for binding to oligodeoxyguanosine.

In mammals scavenger receptors (SR) are expressed by monocytic-macrophage lineage cells and B-cells. Studies of various teleost species have indirectly demonstrated the presence of SR receptors on phagocytic or endothelial cells by showing the uptake of SR ligands (i.e. derivatised (acetylated) lipoproteins) by these cells. In the present study, nonspecific cytotoxic cells (NCC) were examined for membrane expression of an SR-like protein. Approximately 15-25% of purified NCC expressed scavenger receptor class A (SR-A) demonstrated by binding by a monoclonal (2F8) specific for mouse SR-A (types I, II). Flow cytometric analysis determined that SR binding cells had the same size and 'side scatter' characteristics as NCC. Two colour flow analysis of NCC demonstrated that only a subset of NCC expressed the SR-A-like protein and non-NCC were SR-A negative. Membrane expression of SR on NCC was confirmed by fluorescence microscopy. Analysis of the tissue distribution of SR bearing cells demonstrated that in both catfish and tilapia, SR-A was expressed by NCC in the peripheral blood, spleen and anterior kidney. Experiments were also done to determine if the ligands known to bind mammalian SR-A had a similar specificity for the teleost receptor. Cold competition binding experiments determined that anti-SR-A antibody competed with and reduced biotinylated polyguanosine 20-mer binding to NCC by approximately 40%. Two other types of ligands known to bind (mammalian) SR-A (i.e. polyvinyl sulphate and dextran sulphate) likewise decreased anti-SR-A antibody binding to NCC by 40%. These studies for the first time demonstrated that NCC express the teleost orthologue of mammalian SR-A, suggesting that NCC may participate in physiologic regulation of lipid metabolism in addition to functions of innate immunity.

Mechanisms of nonspecific cytotoxic cell regulation of apoptosis: cytokine-like activity of Fas ligand.

The role of FasL/FasR pathways of immunoregulation of programmed cell death in teleost cytotoxic innate immunity has not been previously examined. In the present study, constitutive cytosolic soluble FasL (sFasL) was detected in anterior kidney (AK), peripheral blood (PBL) and liver NCC obtained from tilapia. Ligation of NCC by tumour cells caused the release of sFasL that was associated with lysis of HL-60 targets in 14 h killing assays. Evidence that sFasL mediated this activity was that anti-(human) FasL inhibited tilapia and catfish (cf.) NCC lysis of FasR+ HL-60 tumour cells. Inhibition was concentration dependent. Lysis of IM-9 targets (12% positive for FasR) by (cf.) anterior kidney and PBL NCC was only partially inhibited by anti-FasL mab. Activated NCC from both species were negative for the expression of membrane FasL and FasR. These data confirmed that NCC lyse sensitive tumour cells by multiple effector pathways. Pretreatment of (FasR+) HL-60 cells with anti-FasR mab completely inhibited cf. cytotoxicity at low (100:1) E:T ratios. Anti-FasR mab did not inhibit the lysis of IM-9 targets by cf. NCC. This study demonstrated that for catfish and tilapia, initial target cell conjugate formation was required; however, the terminal killing mechanism depended on at least two different pathways of cytotoxicity. One pathway depended on the release of preformed soluble FasL by activated NCC in the presence of FasR positive target cells. A second pathway has yet to be determined.

The antigen receptor (NCCRP-1) on catfish and zebrafish nonspecific cytotoxic cells belongs to a new gene family characterized by an F-box-associated domain.

The catfish nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. This report identifies and characterizes a zebrafish orthologue of the catfish NCCRP-1. The zebrafish NCCRP-1 cDNA contains an open reading frame that encodes a predicted protein of 237 amino acids with a MW of 27 kDa and a pI of 5.5. Sequence similarities comparisons show that the NCCRP-1 receptors from these two phylogenetically distant species share a high degree of identity. These results suggested that NCCRP-1 performs a crucial function in innate immunity in teleosts. Further, a zebrafish 17-mer peptide corresponding to the catfish NCCRP-1 antigen-binding domain inhibited (catfish) cytotoxicity toward conventional tumor target cells (HL-60). These data appeared to indicate that the zebrafish NCCRP-1 protein may function as an antigen recognition molecule and, as such, may participate in innate immunity in teleosts. A homology search of the zebrafish NCCRP-1 protein revealed that it shares a significant level of identity with another group of proteins belonging to an F-box subfamily. These proteins share an F-box domain in the N terminus (not present in NCCRP-1) and an extremely conserved C-terminal region that has been termed the F-box-associated domain (FBA). The FBA is currently of unknown function. A new gene family is proposed in this work, based on similarities in the FBA sequences with the catfish and zebrafish NCCRP-1 peptides. This new gene family includes several F-box domain-containing proteins and a predicted C. elegans protein.